1. Field of the Invention
This invention generally relates to the field of semiconductor nanocrystals and to the field of cellular behavior and phenomena. More specifically, the invention provides a simple, reliable method of phagokinetic tracking using semiconductor nanocrystals. The invention has further application in the fields of observing live cell motility, migration, metastatic potential, cellular uptake and tracking cell lineage using semiconductor nanocrystals.
2. Description of the Related Art
Metastasis of cells is a major problem in cancer. Migration of cancerous cells leads to metastases and the formation of secondary tumors. Studies of chemotherapeutic agents depend upon cell motility assays. Current assays are cumbersome and prone to error, and require killing the cells which prevents further analyses.
The most direct method for observing cell motility currently is time lapse videos of cells in culture as described by Rajah, et al., In vitro cell. Dev. Biol.—Animal 34, 626-628 (1998). However, this method is restricted to measurements on just a few cells at a time, and therefore this approach is not widely used to make statistically significant studies of cell populations. Improved statistics can be obtained with the “scratched wound method” in which a region of the cell culture substrate is denuded of cells, and then the time scale for the filling of this “hole” is observed; unfortunately, the history of the cell migration paths are lost, and the analysis is complicated by subjective analysis of the complex and variable patterns of the cell motion that lead to hole filling. Környei, et al. J. Neurosci. Res. 61, 421-429 (2000); R. R. Bürk, PNAS 70, 369-371 (1981). A significant advance occurred with the development of the Boyden Chamber invasion assay, in which cells are seeded on one side of a membrane, and the rate of appearance of cells on the other side is monitored. Yao, et al., J. Neurosci. Res. 27, 36-42 (1990); S. Boyden, J. Exp. Med. 115, 453-466 (1962).
There are many commercially available versions of the Boyden Chamber technology. Many companies have modified the Chamber technology to use, for example, a stainless steel chamber (e.g. Neuro Probe BY312 BOYDEN CHAMBER, made by Neuro Probe, Inc., Gaithersburg, Md.) or creating chambers by clamping glass cover slips with silicon spacers (e.g. Hemogenix MODIFIED BOYDEN-CHAMBER #0729.000, made by HemoGenix LLC, Irmo, S.C.), or plastic microplates wherein each of the wells has a coated membrane on the bottom for cells to migrate through (e.g. QCM™ Quantitative Cell Migration Assay, made by CHEMICON International, Inc., Temecula, Calif.). The Boyden Chamber method is by far the most widely used, yet it is laborious; most protocols require that the cells be fixed or stained, and thus destroyed, and do not allow for real time variation of the external condition. Cells can be quantitated through various means such as by optical density or by fluorescence microscopy, but a significant number of cells are frequently lost during processing which decreases accuracy.
A recent cell motility assay has been developed and patented by Biometric Imaging, Inc. See Jarnagin, et al., U.S. Pat. No. 6,238,874. Jarnagin et al. describe an apparatus and method for assaying motility in response to a chemotaxic agent. The apparatus provides a chamber having two regions. The apparatus facilitates the establishment of a concentration gradient of the chemotactic agent which increases on progressing from the first region to the second region, also called the interrogation region. The individual cells are detected by comparing the distribution of detected, position-assigned cells in the interrogation region at two or more time intervals. The method of using this apparatus involves labeling the selected type of cell with a fluorescent compound and detecting the peak fluorescence of individual cells or a population of cells. The positional information for each cell or a population of cells is detected over a period of time by tracking the fluorescence and storing the data as pixel images of coordinates.
Albrecht-Buehler proposed a method for studying cell motility based upon observations of “phagokinetic tracks.” G. Albrecht-Buehler, Cell 12, 333-339 (1977); G. Albrecht-Buehler, Cell 11, 395-404 (1977). In a most general way, a phagokinetic track is generated when a cell passes over a layer of “markers”, and ingests them, leaving behind a blank spot equal to the area the cell has traversed. In principle the method is very powerful, as it provides a rapid and automatic method for integrating cell motility while preserving the history of individual paths. Until now, the method has only been used in a limited way due to problems with the available markers. Markers such as submicron Au (gold) particles, India ink or latex particles have been used because they can be imaged optically using darkfield microscopy. However, these markers also impose many limitations so that the technique has not received widespread acceptance.
The Au particles used in the phagokinetic tracking assay must be large (0.15 microns) in order to be observable optically. Such large particles do not stick well to the substrate, and therefore have to be grown directly on the substrate in a process that yields highly inhomogeneous particle distributions. Since the particles are grown directly on the substrates, by pouring a hot (near boiling) aqueous solution onto the substrate, the range of usable substrates is limited. Further, due to the large size of the Au microcrystals, when a cell moves one diameter, it ingests a volume of Au corresponding to ˜1% of the total cell volume, and there is a strong possibility that this perturbs the cell motility. This is particularly true for small cells, such as epithelial cells, which are of particular importance in studies of metastasis, and in neurons. The film homogeneity is poor, and the inter-particle distance is large, limiting the resolution. Finally, this method provides only one level of contrast, so that it is restricted to measurements in two dimensions. Also it cannot easily be coupled to information about chemical signals involved in cell motility.
Albrecht-Buehler's method of phagokinetic tracking has previously been proposed as an in vitro cancer diagnostic assay. See Zetter, U.S. Pat. No. 4,359,527. The assay involved providing a substrate coated with a layer of visible particles susceptible to ingestion by capillary endothelial cells. Those cells are then seeded onto and allowed to adhere to the substrate. After incubation of the cells with a test sample (usually a fluid such as urine from a patient suspected of having cancer), the visible particle-detected phagokinetic tracks left by the cells are compared to the tracks left by a control sample. If the test tracks are larger, that indicates a positive cancer diagnosis. The track area is measured by fixing the cells and the tracks, projecting the tracks onto a television screen, tracing the projection on a transparent surface and automatically computing the track area on the surface using a digital image analyzer.
“Quantum dots,” referred to herein as “semiconductor nanocrystals,” are protein-sized crystals of inorganic semiconductor nanocrystals, initially developed for opto-electronic applications. Upon excitation by an energy source, semiconductor nanocrystals emit a signal. They are robust and efficient light emitters, with a wide range of potential applications in cell labeling.
The ability to control the growth conditions, shape and size allows one to tailor and control the optical properties of semiconductor nanocrystals. The absorption onset and emissions maximum of semiconductor nanocrystals shift to higher energy with decreasing size. See Bawendi et al., J. Am. Chem. Soc. 115, 8706 (1993). Variations of the material, size and shape used for the semiconductor nanocrystal afford a spectral range of 400 nm to 2 μm in the peak emission, with typical emission widths of 20-30 nm [full width at half maximum (FWHM)] in the visible region of the spectrum and large extinction coefficients in the visible and ultraviolet range. Various sizes of semiconductor nanocrystals can be excited with a single excitation wavelength of light, resulting in the simultaneous detection of multiple emission colors. See Alivisatos et al., Science 281, 2013 (1998).
Because biological applications require water-soluble semiconductor nanocrystals, several methods have been developed to add a solubilizing layer. One strategy relies on covalently coupling a thiolated molecule having a free carboxyl group facing the solution to maintain water solubility. See Nie et al., Science 281, 2016 (1998). However, because the bond holding the thiol to the semiconductor nanocrystal is dynamic, this leads to low stability in water and slow dissolution of the semiconductor nanocrystals and the diffusion. When coated with a suitable solubilizing layer, such as silica, the semiconductor nanocrystals are stable under physiological buffer conditions. See Gerion et al., J. Phys. Chem B 105, 8861-8871 (2001), “Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated CdSe/ZnS Semiconductor Quantum Dots” and Mitchell et al., J. Am. Chem. Soc., 121 (35), 8122-8123, 1999. These semiconductor nanocrystals maintain their optical properties and are soluble in solutions over a wide range of pH.
The silanization method to provide solubility to semiconductor nanocrystals is fully described in Gerion et al, J. Phys. Chem B 105, 8861-8871 (2001) and illustrated in FIG. 1 of the same reference. The method relies upon the siloxane bond (—Si—O—Si—) in the following reaction, wherein each Si atom is actually bound to three methoxy groups per molecule and one residual group, but the reaction shown only describes the reaction of one of the methoxy groups.R1R2R3—Si—OCH3+H2O→R1R2R3—Si—OH+CH3OHR1R2R3—Si—OH+HO—Si—R1R2R3→R1R2R3—Si—O—Si—R1R2R3+H2O
Limitations on biological markers have prompted researchers to use semiconductor nanocrystals as fluorescent biological labels. See Bruchez et al., Science 281, 2013-2016 (1998). Others have developed “quantum dot” bioconjugates for detection by coupling luminescent semiconductor nanocrystals to biological molecules. For example, see Chan et al., Science 281, 2016-2018 (1998) and Bruchez et al., Science 281, 2013-2016 (1998). Hydroxylated “quantum dots” have recently been used as luminescent probes for in situ hybridization as demonstrated by Pathak et al., J. Am. Chem. Soc. 123, 4103-4104 (2001). “Quantum dots” have also been used to tag microbeads for multiplexed optical coding of biomolecules by embedding different-sized ZnS-capped CdSe semiconductor nanocrystals into polymeric microbeads. See Nie et al., Nature Biotechnology 19, 631-635 (2001).
Until now, “quantum dot” based biological labeling experiments have been confined to static labeling. Prior publications and usage of “quantum dots” in biological applications have been limited to these bioconjugate “quantum dots”, where the semiconductor nanocrystals were decorated with proteins, antibodies, nucleic acids, oligonucleotides and other organo- or affinity molecules as hybridization probes in fluorescence assays or to mediate receptor-mediated endocytosis. Prior work using decorated semiconductor nanocrystals have all relied upon the attached biomolecules for entry into cells or for use in a specific biological application and have not attempted to use undecorated “quantum dots.” See Alivisatos et al., U.S. Pat. No. 6,207,392.